Homogeneous fluorescence quantitative detection method for base deletion site in genomic DNA sequence

Abstract

The application discloses a homogeneous fluorescence quantitative detection method for base deletion sites in genomic DNA sequences, and covalently labels known sequence oligonucleotide chains to the base deletion sites through a covalent connection reaction, and realizes quantitative detection of the base deletion sites in the genomic DNA sequences by using Lambda exonuclease and corresponding fluorescence probes. The application is simple in operation, high in sensitivity, small in required biological sample amount, and can conveniently, quickly, sensitively and accurately determine the content of the base deletion sites in biological samples, and has high application value in biomedical research, clinical diagnosis and drug development evaluation related to DNA damage and repair.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analysis and detection of genome apurinic / apyrimidinic sites (AP sites for short), and more particularly to a kind of exonuclease-oligonucleotide fluorescent probe combined reaction system, and a method for quantitatively detecting the content of AP sites in cell lysate, blood cell liquid and the like samples by using the fluorescent probe. BACKGROUND

[0002] Apurinic / apyrimidinic sites (AP sites for short) are a kind of common genomic DNA damage. AP sites are mainly produced by spontaneous glycosidic bond hydrolysis in cells. After losing the base, only the sugar ring and the phosphate backbone structure are left. It is estimated that 10,000-50,000 AP sites are spontaneously hydrolyzed in each human cell per day under physiological conditions. In addition, base excision repair processes and some exogenous damage factors such as ultraviolet light, ionizing radiation or environmental acidification can also cause different degrees of base loss and form AP sites.

[0003] Since AP sites lose the key genetic information of the base structure, they will hinder DNA replication and transcription. If not repaired in time, they will produce cell toxicity, induce genetic mutations, and cause cell apoptosis. Base excision repair (BER) is the main repair pathway for AP sites and other types of base damage. AP sites are not only a product of direct DNA damage, but also an important intermediate in the base excision repair process of other types of base damage. The content level and sequence distribution of AP sites in the genomic DNA in cells are closely related to aging and the occurrence and development of various diseases, especially cancer. It has been found that the content of AP sites in cells of patients with Parkinson's syndrome, Alzheimer's disease and some cancers is higher. On the other hand, there are also many reports on the method of cancer treatment by inducing a large number of base damage in cancer cells and modifying the important intermediate AP sites in BER to inhibit damage repair. Therefore, quantitative determination of the content of AP sites in the genomic DNA of cells is helpful for monitoring the health status of the human body, evaluating the effect of drug treatment, and also has a positive promoting effect on in-depth study of important influencing factors of BER process and discovery of new drug targets.

[0004] Compared with DNA base changes such as gene mutation, the quantitative detection of abasic sites faces the following three challenges: (1) the content of abasic sites is low, which cannot be amplified by PCR; (2) new abasic sites are easily introduced during sample processing; (3) abasic sites have no characteristic metabolites. Therefore, the detection method of abasic sites needs to have the basic characteristics of mildness, sensitivity, good specificity, accuracy and reliability. The commonly used quantitative analysis methods for abasic sites are aldehyde reactive probe-enzyme linked immunosorbent assay (ARP-ELISA) and liquid chromatography-mass spectrometry (HPLC-MS) methods. Both of these two methods need relatively complicated operation steps, which are time-consuming and laborious. The ARP-ELISA method covalently connects the hydroxylamine group of ARP to the aldehyde group of AP site, and labels biotin to the AP site, and then quantitatively detects it by streptavidin-horseradish peroxidase according to the ELISA-like method. This method can detect 1-40 AP sites / 10 5 Base pairs in 1 μg of DNA sample, which has low detection sensitivity and high sample demand, and the ARP-ELISA method cannot meet the needs of actual biological sample detection. It is worth noting that in addition to the covalent connection between the hydroxylamine group and the AP site, other aldehyde-modified bases such as 5-aldehyde cytosine (5fC) and 5-aldehyde uracil (5fU) in DNA can also covalently connect with ARP, and then be detected together by subsequent steps, thereby bringing false positive signals. Since the content of these aldehyde-modified base structures in cells is comparable to that of AP sites, the interference cannot be ignored. In addition, the ARP-ELISA method has poor repeatability, and the single detection time is as long as about 30 hours. The principle of HPLC-MS method for detecting AP sites is to first label the probe with characteristic mass spectrometry response on the AP site through the hydroxylamine group, and then hydrolyze the DNA chain into single nucleotides by a series of enzymes, and determine the purified enzyme products by mass spectrometry. This method can detect 2.2 AP sites / 10 8 Nucleotides, which can distinguish AP sites from other aldehyde-containing base structures by mass spectrometry identification, can avoid introducing false positive signals. The disadvantage of this method is that the pretreatment step is complex and cumbersome, the efficiency of the enzyme digestion and purification steps is difficult to guarantee, and the DNA sample demand for single detection is as high as 5-13.5 μg, and the detection time is about 24 hours. Therefore, it is urgent to develop a simple, rapid, highly sensitive, selective, and reproducible AP site quantitative detection method to realize accurate and reliable and high-throughput detection of AP site content in biological samples (such as cell lysates, blood cell liquids, etc.). SUMMARY

[0005] The present application aims to provide a simple, rapid, sensitive, small sample requirement, accurate and reliable analysis method for detecting AP site content in biological samples (cultured cells, human blood cells, tissue cells, etc.), so as to overcome the limitations in the prior art.

[0006] The technical solution and detection principle of the present application are shown in Figure 1 The hydroxylamine group at the 5' end of the oligonucleotide chain X is modified, and the AP site converted into an aldehyde group structure at the 5' end by T4 PDG (an AP site cleavage enzyme) is covalently labeled. The auxiliary chain Y is added to form a double-stranded DNA structure with 2 nucleotide mismatches at the 5' end of the X chain and the rest matched. The excess X chain not reacted with the AP site is hydrolyzed and removed by Lambda exonuclease. Then the probe chain P modified with 5'-FAM and 3'-BHQ1 is added to hybridize with the X chain labeled on the AP site to form a double-stranded DNA structure with 2 nucleotide mismatches at the 5' end of the P chain and the rest matched. Under the action of Lambda exonuclease, the P chain is rapidly hydrolyzed to emit a fluorescence signal, and the released X chain labeled on the AP site continues to hybridize with the remaining P chain to realize amplification of the fluorescence signal. The content of AP site in the sample is accurately quantified by detecting the rate of fluorescence signal rising in the reaction solution.

[0007] The technical solution of the present application is as follows:

[0008] A quantitative detection method for base deletion sites in genomic DNA sequences, comprising the following steps:

[0009] 1) preparing an oligodeoxynucleotide chain with a hydroxylamine modification at the 5' end, i.e. X chain;

[0010] 2) treating the base deletion site in the genomic DNA sequence with T4 PDG to hydrolyze it into an aldehyde group structure at the 5' end, and then adding the X chain for covalent labeling reaction;

[0011] 3) after the covalent labeling reaction is completed, the auxiliary chain Y and the excess X chain not reacted with the base deletion site are hybridized, the Y chain hybridizes with the free X chain to form a double-stranded DNA structure with 2 nucleotide mismatches at the 5' end of the X chain and the rest matched, the excess X chain is hydrolyzed and removed by Lambda exonuclease, and then heated to inactivate the Lambda exonuclease;

[0012] 4) adding P strand hybridization to the reaction product solution of step 3) which is an oligonucleotide probe labeled with a fluorescent group and a quencher group at the 5' and 3' ends respectively, hybridizing with the X strand covalently labeled at the abasic site to form a double-stranded DNA structure with 2 nucleotide mismatches at the 5' end of the P strand and the rest of the structure matched, then adding Lambda exonuclease, the P strand is quickly hydrolyzed to emit a fluorescent signal, and the fluorescent signal is collected in real time;

[0013] 5) accurately quantifying the content of the abasic site in the sample by detecting the rate of the fluorescent signal rising in the reaction solution.

[0014] The length of the X strand in step 1) is 14-16 nt, preferably 14 nt.

[0015] The X strand in step 1) is obtained by the alkyne-azide cycloaddition click chemistry reaction of the oligodeoxynucleotide chain with the azide group modified on the 5' end phosphate backbone and N-(propargyloxy)phthalimide under the catalysis of copper sulfate / sodium ascorbate. Preferably, the buffer solution of the alkyne-azide cycloaddition click chemistry reaction is a phosphate buffer, and a ligand tris-(3-hydroxypropyltriazolylmethyl)amine is added to improve the catalytic efficiency of copper.

[0016] In step 2), T4 PDG with AP lyase activity is used to enzymatically hydrolyze the AP site into an α, β-unsaturated aldehyde structure, and then the X strand is added for covalent labeling. Preferably, the X strand is added after the T4 PDG enzymatic reaction, and the covalent labeling reaction is carried out in a phosphate buffer at room temperature for 1.5-2 hours.

[0017] In step 3), Y strand and Lambda exonuclease reaction buffer are first added, mixed thoroughly, and then allowed to stand to hybridize the Y strand with the X strand, then Lambda exonuclease is added for incubation for a period of time, and finally heated to 90°C to inactivate the Lambda exonuclease.

[0018] Preferably, the P strand in step 4) is labeled with a fluorescent group at the 5' end and a quencher group at the 3' end. The fluorescent group and the corresponding quencher group are preferably one of the following combinations: FAM and BHQ1 / DABCYL / TAMRA, TET and BHQ1 / DABCYL, HEX and BHQ1 / BHQ2 / DABCYL, TAMRA and BHQ2, ROX and BHQ2, Texas Red and BHQ2, Cy5 and BHQ2 / BHQ3.

[0019] In the quantitative detection method of the base deletion site in the genomic DNA sequence, the fluorescence rising rate of the sample to be detected is measured by the curve of fluorescence intensity changing with time collected in step 4), and the content of the base deletion site in the sample to be detected is calculated according to the standard curve obtained by linear fitting of the fluorescence rising rate and the AP site concentration of the standard sample in step 5).

[0020] According to the above method, the application further provides a quantitative detection kit for a base deletion site in a genomic DNA sequence, comprising three oligonucleotide chains: X chain, Y chain and P chain, T4 PDG enzyme and its working buffer, Lambda exonuclease and its working buffer; wherein the X chain is an oligodeoxynucleotide chain with a hydroxylamine modification at the 5' end; the Y chain can hybridize with the X chain to form a double-stranded DNA structure with 2 mismatched nucleotides at the 5' end of the X chain and the remaining part matched; and the P chain is an oligonucleotide probe labeled with a fluorescent group and a quenching group at both ends, which can hybridize with the X chain to form a double-stranded DNA structure with 2 mismatched nucleotides at the 5' end of the P chain and the remaining part matched.

[0021] Compared with the prior art, the application has the following outstanding advantages:

[0022] (1) High selectivity. The application uses an oligonucleotide sequence (X chain) with a 5' end hydroxylamine modification and a length of 14-16 nt to label the AP site completely converted into a terminal aldehyde structure by T4 PDG AP cleavage enzyme through a covalent reaction, which has three important contributions, one is that the reaction efficiency of the terminal aldehyde group is significantly improved than the intrachain hemiacetal before conversion, which ensures the labeling efficiency of the AP site; two is that the steric hindrance caused by the oligonucleotide sequence with a length of 14-16 nt makes it difficult for the modified aldehyde group on the base, such as 5fC and 5fU, to participate in the reaction, and the labeling rate is greatly reduced; three is that after the 5' end is labeled to the AP site, it is embedded in the middle of the chain, which is structurally different from the remaining X chain that has not reacted, laying a foundation for the subsequent step of removing the excess X chain.

[0023] (2)High sensitivity, small sample requirement. After the 14-16 nt long X strand is labeled to the AP site, the X strand can be detected by the method (Tongbo Wu, Wei Chen, Ziyu Yang, Haocheng Tan, Jiayu Wang, Xianjin Xiao, Mengyuan Li and Meiping Zhao, DNA terminal structure-mediated enzymatic reaction for ultra-sensitive discrimination of single nucleotide variations in circulating cell-free DNA, Nucleic Acids Research, 2018, 46(4), e24) reported by the research group before, which uses Lambda exonuclease combined with 5'-FAM labeled fluorescent probe to amplify the fluorescence signal of the X strand. This strategy effectively solves the problem that the AP site cannot be amplified by PCR, greatly improves the detection sensitivity, and significantly reduces the sample requirement. This is of great significance to the precious and limited biological samples to be tested.

[0024] (3)Simple operation, short time consumption. The AP cleavage enzyme reaction and the covalent labeling reaction of the X strand to the AP site after enzyme hydrolysis in the method are simple and rapid, and the subsequent fluorescence signal amplification reaction can also be quickly completed. The key rate-determining step is the removal of the excess X strand. In the present application, we determined by mass spectrometry that when the free X strand hybridizes with the auxiliary Y strand to form a double strand, as long as the 5' hydroxylamine of the X strand is the exposed end and the two adjacent nucleotides are opened due to the mismatch with the Y strand, the end can be recognized and hydrolyzed by Lambda exonuclease (see Example 1 for details). The X strand covalently labeled to the AP site is not affected because its 5' end is closed in the product DNA strand. This ingenious design makes the entire detection process continuous in the same homogeneous solution without the need for separation, purification and other cumbersome steps. A single determination only takes 5 hours, which is significantly faster than the existing methods and can be used for rapid detection of large quantities of samples.

[0025] Since AP site is a key intermediate of BER process, for other types of base damage, the damaged base can be removed by adding corresponding glycosylase first, converted into AP site, and then the AP site is quantified by the method of the present application, so as to indirectly quantify various types of base damage. The present application is simple in operation, high in sensitivity, small in required biological sample amount, and can conveniently, quickly, sensitively and accurately determine the content of AP site in a biological sample, and has high application value in biomedical research, clinical diagnosis and drug development evaluation related to DNA damage and repair. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 . The principle diagram of the present application for quantitatively detecting the content of AP site by homogeneous fluorescence analysis method.

[0027] Figure 2 . The experiment of preparing 5' end hydroxylamine modified oligonucleotide chain X and covalently labeling AP site by X in Example 1 of the present application, wherein: (a) the reaction formula of the preparation reaction of hydroxylamine modified X chain; (b) the mass spectrum of the hydroxylamine modified X chain, the theoretical molecular weight of X chain is Mw=4591.3 Da; (c) the gel electrophoresis analysis result of the covalent labeling product of X to AP site.

[0028] Figure 3 . The mass spectrum analysis result of the hydrolysis product of 5' end hydroxylamine modified X chain assisted by Y chain in Example 1 of the present application, the product peak with a molecular weight of 611.25 Da in the figure shows that the 5' end modified hydroxylamine-triazole ring of X chain and the first 5' end nucleotide connected thereto are completely removed, and the generated 5' phosphate end can make the hydrolysis reaction continue, so as to remove the free X chain.

[0029] Figure 4 . The experimental result of detection sensitivity in Example 3 of the present application, wherein: (a) the fluorescence response curve corresponding to different concentrations of AP site standard sample; (b) the standard curve and linear fitting result (n=3).

[0030] Figure 5 . The schematic diagram of cell culture process and group number of extracted cell genomic samples at different time points in Example 4 of the present application, wherein group A represents a control group without damage treatment, group B is treated by MMS (methyl damage), and group C is treated by t-BHP (oxidation damage). DETAILED DESCRIPTION

[0031] The present application will be further described below by specific examples. Those skilled in the art should understand that these examples are only used to illustrate the present application and not to limit the scope of the present application.

[0032] The nucleic acid sequences involved in the experiment are shown in Table 1, and the components of the nuclease and its working buffer are shown in Table 2.

[0033] Table 1. Nucleic acid sequences used in the present application

[0034]

[0035] 1 FAM: 6-carboxy-fluorescein (6-carboxy-fluorescein); BHQ1: Black hole quencher 1; 2 X represents an AP site.

[0036] Table 2. Nucleic acid enzymes and their working buffers

[0037]

[0038]

[0039] Example 1 Covalent labeling of AP site by 5' end hydroxylamine modified DNA strand and removal of the remaining strand

[0040] (1) Preparation of 5' end hydroxylamine modified DNA strand (X strand, sequence shown in Table 1)

[0041] In this work, N-(propargyloxy)phthalimide with protected hydroxylamine group was used as raw material, and oligodeoxynucleotide chain X-N3 (sequence shown in Table 1) with azide group modified on the 5' end phosphate backbone was used as raw material. The preparation reaction of hydroxylamine modified X chain was completed by alkyne-azide cycloaddition click chemistry reaction under the catalysis of copper sulfate / sodium ascorbate (see Figure 2 The specific operation steps are as follows: in a total volume of 600 μL reaction system, water, 60 μL 10×phosphate buffer solution, X-N3 with a final concentration of 5 μM, 500 μM N-(propargyloxy)phthalimide, 625 μM tris-(3-hydroxypropyltriazolylmethyl) amine, 125 μM copper sulfate, and 625 μM sodium ascorbate were added, and then stirred uniformly. After reaction at room temperature for 4 hours, the reaction system was ultrafiltered to remove excess small molecules by using 3K ultrafiltration tube, and the operation was repeated for 3 times. Subsequently, the nucleic acid containing solution was diluted to 185 μL with water, and 15 μL of hydrazine monohydrate (mass fraction 70%) was added, and then reacted at room temperature for 40 minutes. The nucleic acid was purified by using Oligo Clean & Concentrator kit. In order to avoid the combination of copper and DNA strands and the influence on subsequent detection, ligand tris-(3-hydroxypropyltriazolylmethyl) amine was added to improve the catalytic efficiency of copper. 2+ Figure 2 ​As shown in (b), the reaction successfully obtained an oligodeoxynucleotide chain with a 5' end modified with hydroxylamine, i.e., X chain.

[0042] (2) Covalent labeling reaction of X chain to AP site and removal of excess X chain

[0043] The 68bp double-stranded DNA containing U bases composed of sequences 68-U and 68-C (sequences are shown in Table 1) was used as the substrate. After treatment with uracil-DNA glycosylase (UDG), the standard nucleic acid sample containing AP sites was obtained. T4 PDG with AP lyase activity was added to enzymatically cleave the AP sites into a, β-unsaturated aldehyde structure, and then X chain was added for covalent labeling. The specific operation steps are as follows: 5μM of 68-C and 68-U, UDG reaction buffer and water were added to a 200μL PCR tube, and the solution was heated to 90℃ and then gradually cooled to 25℃. A total of 5U of UDG was added to a final reaction volume of 50μL. Incubation was performed at 37℃ for 2 hours. After the reaction, the nucleic acid was purified using the Oligo Clean & Concentrator kit. In a 200μL PCR tube, 150nM of the purified product, 2μL of 10×phosphate buffer solution, and a total of 5U of T4 PDG were added to a final reaction volume of 20μL. Incubation was performed at 37℃ for 30 minutes, at which time all AP sites were cleaved into aldehyde end structures. Subsequently, 20μM of X chain, 1μL of 10×phosphate buffer solution and water were added to a final reaction volume of 30μL, and the reaction was performed at room temperature for 2 hours. After the reaction, the DNA standard that did not participate in the ligation reaction was used as a control, and the product was characterized by non-denaturing polyacrylamide gel electrophoresis. As shown in (b), the X chain was successfully labeled on the AP site. Figure 2 As shown in (c), the X chain was successfully labeled on the AP site.

[0044] Further, by adding the auxiliary chain Y chain (sequence is shown in Table 1) and Lambda exonuclease (buffer composition is shown in Table 2), the excess X chain that did not react with the AP site was hydrolyzed and removed. The specific operation steps are as follows: In a 200μL PCR tube, 5μL of the above covalent labeling reaction product solution, 10nM of Y chain, 4μL of Lambda exonuclease reaction buffer, and water were added to a final volume of 40μL. After mixing well, the Y chain was hybridized with the X chain by standing for 10 minutes. A final concentration of 300U / mL of Lambda exonuclease was added and incubated at 37℃ for 30 minutes. Then, heating to 90℃ for 10 minutes inactivated the Lambda exonuclease.

[0045] This step reaction takes advantage of an important property discovered for the first time by the present application and confirmed by mass spectrometry, that is, when the X strand obtained after modification of the 5' end by the triazole ring formed by the cycloaddition of hydroxylamine to the alkyne-azide and the connecting arm, when hybridized with the auxiliary Y strand to form a double strand, as long as the 5' hydroxylamine of the X strand is the exposed end and the two adjacent nucleotides are open due to mismatch, Lambda exonuclease can completely remove the 5' end modification of the hydroxylamine-triazole ring and the 5' end first nucleotide connected to it, and the 5' phosphate end produced allows the hydrolysis reaction to continue, thereby removing the free X strand. The reaction product was identified by mass spectrometry, and the results are shown in Figure 3 This ingenious design and efficient removal capability provide a breakthrough technical improvement for simplifying the entire detection process. The product does not require other separation and purification steps, and can be directly linked to the subsequent fluorescence signal amplification detection reaction.

[0046] Example 2 Selectivity study of the method established by the present application

[0047] We synthesized dsDNA standard S1 containing a single AP site, dsDNA standard S2 containing a single 5fC modification site, and dsDNA standard S3 containing a single 5fU modification site, respectively, to control the efficiency and selectivity of the covalent labeling reaction of the X strand. The specific steps are as follows:

[0048] (1) In a 200 μL PCR tube, add dsDNA standard (S1, S2 or S3) with a final concentration of 2 nM, 0.4 μM X strand, 5 U of T4 PDG, and phosphate buffer solution to make the final volume of the solution 15 μL, and incubate at room temperature for 2 hours. Take 5 μL of the product solution and add it to another PCR tube, then add 4 μL of 10x Lambda working buffer, 10 nM of Y strand, and water to make the final volume 40 μL. Mix well and then add Lambda exonuclease with a final concentration of 300 U / mL, and incubate at room temperature for 30 minutes. Heat to 90°C for 10 minutes to inactivate the Lambda exonuclease.

[0049] (2) Add water, a final concentration of 300nM P chain, and 1μL 10×Lambda working buffer to the final product solution of step (1), mix well, and perform a temperature rise annealing step in a real-time fluorescence PCR instrument. Add a final concentration of 250U / mL Lambda exonuclease to make the total volume of the solution 50μL (the final concentration of each standard sample is 200pM at this time), and use a real-time fluorescence PCR instrument to collect fluorescence data at 37°C. The instrument used for detection is a Rotor-Gene Q real-time fluorescence PCR instrument, the detection channel is the Green channel, the excitation wavelength is 470±10nm, the detection wavelength is 510±5nm, and the gain value is 10. The program setting for the temperature rise annealing step is 95℃ (90s)–75℃ (90s)–55℃ (90s)–25℃ (90s). When detecting the fluorescence intensity at 37°C, the fluorescence intensity is read every 5s.

[0050] As the reaction proceeds, the fluorescence signal of the solution begins to rise. Based on the fluorescence intensity versus time curve, the slope of the linearly rising portion of the fluorescence curve is the fluorescence rise rate, representing the level of labeled X chain in the corresponding standard sample. Using the fluorescence rise rate of the reaction solution of standard S1, which addresses the AP site, as a 100% control, the test results show that the fluorescence rise rates of the reaction solutions of standards S2 (5fC) and S3 (5fU) are 1.7% and 13.0%, respectively. This indicates that the labeling efficiency of X chain at the AP site is far greater than that for 5fC and 5fU, demonstrating that the method of the present invention has good selectivity for the AP site.

[0051] Example 3 <Working Curve and Sensitivity of AP Site Detection Method of the Present Invention>

[0052] The complete process of detecting AP sites by the method of the present invention is as follows:

[0053] Step 1: Covalent labeling of AP sites in the standard or test sample

[0054] The reaction was performed in a 200 μL PCR tube, maintaining a final reaction volume of 15 μL. 1.5 μL of 10× phosphate buffer and AP site standards or test samples at final concentrations of 2 nM, 1 nM, 500 pM, 200 pM, 50 pM, and 25 pM were added to the system. A blank control was used without the addition of standard or sample. After each solution was mixed thoroughly, a total of 5 U of T4 PDG and a final concentration of 0.4 μM of X chain were added. Water was added to the volume, mixed thoroughly, and incubated at 37°C for 2 hours. The 15 μL ligation product was divided into three replicates for subsequent detection steps.

[0055] Step 2: Use the Y chain to remove the excess X chain

[0056] The reaction was carried out in a 200 μL PCR tube, and the final reaction volume was kept at 40 μL. 4 μL Lambda exonuclease reaction buffer, 5 μL ligation reaction product solution from step 1 and Y strand with a final concentration of 10 nM were added into the system, and the solution was mixed thoroughly and left for 10 minutes to promote the hybridization of Y strand and X strand. Then, 300 U / mL Lambda exonuclease was added, and the solution was incubated at 37 °C for 30 minutes. The solution was heated to 90 °C for 10 minutes to inactivate the Lambda exonuclease.

[0057] Step 3: Determination of AP site content

[0058] To the reaction product solution from step 2, P strand with a final concentration of 300 nM and 1 μL Lambda exonuclease reaction buffer were added, and the solution was heated to 90 °C and then gradually cooled to 25 °C. The total volume of the solution was kept at 50 μL by adding 250 U / mL Lambda exonuclease, and the fluorescence data were collected using a real-time fluorescence PCR instrument at 37 °C. The excitation wavelength was 470 ± 10 nm, the detection wavelength was 510 ± 5 nm, the gain value was 10, and the fluorescence intensity was read every 5 seconds. The fluorescence intensity-time curve (see Fig. 2(a)) was used to determine the fluorescence rising rate, and the linear fitting was performed between the fluorescence rising rate and the final concentration of the abasic site in the standard sample. The working curve is shown in Fig. 2(b). The linear range of the detection method of the present application was 2.5 pM to 200 pM, and the lowest detectable concentration was 2.5 pM. Figure 4 Figure 4 Step 3: Determination of AP site content

[0059] Example 4: Detection of AP site content in different types of cells under different damage-repair conditions by the method developed in the present application

[0060] Three different types of cells, A549, HeLa and MCF-7, were treated with tert-Butylhydroperoxide (t-BHP) or Methyl methanesulphonate (MMS), two different damage reagents. The AP site content in the genome of the cells before and after damage and 18 hours after repair was detected. The cell culture process and sample grouping are shown in Fig. 1. Figure 5 ​MMS, 250 μM t-BHP and no damage reagent control for 1 hour, and the corresponding genomic DNA of the cells was extracted and labeled as MCF-7 cell groups B0, C0 and A0, respectively. A549 cells were further introduced into the cell damage repair process. Half of the cells were cultured in three groups of medium containing 200 μM MMS, 250 μM t-BHP and no damage reagent control for 1 hour, and the corresponding genomic DNA of the cells was extracted and labeled as A549 cell groups B0, C0 and A0, respectively. The other half of the damaged cells were cultured in normal medium without damage reagent for 18 hours for damage repair, and then the corresponding genomic DNA of the cells was extracted and labeled as A549 cell groups B18 and C18. The treatment process of HeLa cells was similar to that of A549 cells. Half of the cells without damage reagent control were cultured for 18 hours for further control, which was labeled as HeLa cell group A0. The remaining treatment and numbering steps were the same as those of A549 cells.

[0061] The specific detection steps are as follows:

[0062] (1) The reaction was carried out in a 200 μL PCR tube, and the final total volume was maintained at 18 μL. 2 μL of Fragmentase, enzyme reaction buffer solution and no more than 3 μg of the genomic DNA to be detected were added, and water was added to make up the volume, fully mixed and incubated at 37°C for 15 minutes. 5 μL of 0.5M EDTA solution was added to terminate the reaction, and the product was extracted and purified by DNA Clean & Concentrator, and then quantified for step (2).

[0063] (2) The reaction was carried out in a 200 μL PCR tube, and the final total volume was maintained at 15 μL. 1.5 μL of 10x phosphate buffer solution, AP site standard sample with final concentrations of 2nM, 1nM, 500pM, 200pM, 50pM, 25pM (used to draw the working curve) or 0.5-1.5 μg of the genomic DNA sample treated in step (1) were added, and the reaction buffer without the standard was used as a blank control. After mixing, 5U of T4 PDG and 0.4 μM of X chain were added, and water was added to make up the volume, and then incubated at room temperature for 2 hours.

[0064] (3) The solution obtained in step (2) was divided into three equal parts of 5 μL each, 4 μL of Lambda exonuclease reaction buffer, 10 nM of Y chain and water were added to make the total volume 40 μL, and then fully mixed. 300 U / mL of Lambda exonuclease was added and incubated at 37°C for 30 minutes. The system was heated to 90°C for 10 minutes to inactivate the original Lambda exonuclease.

[0065] (4) In the reaction solution of step (3), water, P strand with a final concentration of 300 nM, 1 μL Lambda exonuclease reaction buffer were added, and after mixing evenly, the temperature was raised for annealing. Lambda exonuclease with a final concentration of 250 U / mL was added to keep the total volume of the solution at 50 μL, and the fluorescence data were collected by using a real-time fluorescence PCR instrument at 37 °C.

[0066] In this example, the detection instrument used in detection was Rotor-Gene Q real-time fluorescence PCR instrument, the detection channel was Green channel, the excitation wavelength was 470 ± 10 nm, the detection wavelength was 510 ± 5 nm, and the gain value was 10. The program setting of the temperature annealing step was 95 °C (90 s) - 75 °C (90 s) - 55 °C (90 s) - 25 °C (90 s), and the fluorescence intensity was read every 5 s when the fluorescence intensity was detected at 37 °C. The detection results are listed in Table 3.

[0067] Table 3. AP site content determination results of three different types of cells under different damage-repair conditions (AP / 10 6 nt)

[0068]

[0069] / represents undetected

[0070] The cell culture process and the time point of extracting the genomic sample of the cell represented by the group number in Table 3 are shown in Figure 5 For example, group B, B0 represents the genomic DNA extracted immediately after damage treatment, and B18 represents the genomic DNA extracted after 18 hours of natural repair after stopping damage and replacing the normal culture medium. The results in Table 3 show that after being subjected to methylation damage and oxidative damage, the content of the AP site of the three types of cells is significantly increased. After stopping damage and undergoing 18 hours of repair, the content of the AP site is decreased. To determine the recovery rate, in step (2), 100 pM of double-stranded DNA standard S1 containing a single AP site (the sequence is shown in Table 1) was added to each sample to be tested, and the other steps were the same. The measured recovery rate of A549-A0 group was 121 ± 14% (n = 2), and the recovery rate of HeLa-C18 group was 113 ± 7% (n = 2). It is shown that the results measured by the method of the present application are accurate and reliable.

[0071] In addition, the AP site content in the blood cell liquid of a healthy volunteer was also detected by using the method of the present application. The content level of the AP site was 1.3 ± 0.6 AP sites / 10 7 nt, which is lower than the AP site content in the cultured cells. SEQUENCE LISTING <110> Peking University <120> Homogeneous fluorescent quantitative detection method for base deletion site in genomic DNA sequence <130> WX2021-03-190 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 14 <212> DNA <213> Artificial sequence <400> 1 atgtcgatcc gtct 14 <210> 2 <211> 17 <212> DNA <213> Artificial sequence <400> 2 ctaagacgga tcgacct 17 <210> 3 <211> 14 <212> DNA <213> Artificial sequence <400> 3 atacggatcg acat 14 <210> 4 <211> 68 <212> DNA <213> Artificial sequence <400> 4 gtatggtgta cacatgtatg tctacatcac tutatgctgc actgctgtat gtacagtact 60 gtatgcat 68 <210> 5 <211> 68 <212> DNA <213> Artificial sequence <400> 5 atgcatacag tactgtacat acagcagtgc agcataaagt gatgtagaca tacatgtgta 60 caccatac 68 <210> 6 <211> 68 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (32)..(32) <223> AP site <400> 6 gtatggtgta cacatgtatg tctacatcac txtatgctgc actgctgtat gtacagtact 60 gtatgcat 68 <210> 7 <211> 68 <212> DNA <213> Artificial Sequence <400> 7 atgcatacag tactgtacat acagcagtgc agcataaagt gatgtagaca tacatgtgta 60 caccatac 68 <210> 8 <211> 99 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (27)..(27) <223> 5-formylcytosine <400> 8 tcgcacacgc tcagtcaggt agagatctag gagggtggag aggtggttgg agagggttag 60 gaggaagagt gaggtagtga gagggtggag gtgagtgag 99 <210> 9 <211> 99 <212> DNA <213> Artificial Sequence <400> 9 ctcactcacc tccaccctct cactacctca ctcttcctcc taaccctctc caaccacctc 60 tccaccctcc tagatctcta cctgactgag cgtgtgcga 99 <210> 10 <211> 99 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (71)..(71) <223> 5-formyluracil <400> 10 tagccatact gcctcgtccg gacacgacag gaggaaagcc aagacacacg aaccaagaga 60 accaagcaag ucagaagagc acaagcagac cagcgaaca 99 <210> 11 <211> 99 <212> DNA <213> Artificial Sequence <400> 11 tgttcgctgg tctgcttgtg ctcttctgac ttgcttggtt ctcttggttc gtgtgtcttg 60 gctttcctcc tgtcgtgtcc ggacgaggca gtatggcta 99

Claims

1. A method for quantitatively detecting base deletion sites in genomic DNA sequences for non-disease diagnosis purposes, comprising the following steps: 1) preparing an oligodeoxynucleotide chain with a hydroxylamine-modified 5' end, i.e. X chain, the length of the X chain being 14-16 nt; 2) enzymatically digesting base deletion sites in genomic DNA sequences into 5' end-exposed aldehyde groups by T4 PDG treatment, and then adding the X chain for covalent labeling reaction; 3) after the covalent labeling reaction, adding an auxiliary chain Y chain and excess X chain that has not reacted with the base deletion sites for hybridization, the Y chain hybridizing with the free X chain to form a double-stranded DNA structure with 2 nucleotide mismatches at the 5' end of the X chain and the rest being matched, and then adding Lambda exonuclease to hydrolyze and remove the excess X chain, and then heating to inactivate the Lambda exonuclease; 4) adding a P chain for hybridization in the reaction product solution obtained by removing the excess X chain in step 3), the P chain being an oligonucleotide probe labeled with a fluorescent group and a quenching group at both ends, hybridizing with the X chain covalently labeled at the base deletion sites to form a double-stranded DNA structure with 2 nucleotide mismatches at the 5' end of the P chain and the rest being matched, and then adding Lambda exonuclease, the P chain being rapidly hydrolyzed to emit a fluorescent signal, and real-time acquisition of the fluorescent signal; 5) accurately quantifying the content of base deletion sites in the sample by detecting the rate of increase of the fluorescent signal of the reaction solution.

2. The quantitative detection method according to claim 1, wherein In step 1, the oligodeoxynucleotide chain with an azide group-modified 5' phosphate backbone and N-(propargyloxy)phthalimide are subjected to click chemistry reaction of alkyne-azide cycloaddition under the catalysis of copper sulfate / sodium ascorbate to obtain the X chain.

3. The quantitative detection method according to claim 1, wherein In step 1, the buffer solution for the click chemistry reaction of alkyne-azide cycloaddition is a phosphate buffer, and a ligand tris-(3-hydroxypropyltriazolylmethyl)amine is added to improve the catalytic efficiency of copper.

4. The quantitative detection method according to claim 1, wherein In step 2, the X chain is added after the T4 PDG enzymatic digestion reaction, and the covalent labeling reaction is carried out at room temperature in a phosphate buffer for 1.5-2 hours.

5. The quantitative detection method according to claim 1, wherein In step 3, the Y chain and Lambda exonuclease reaction buffer are first added, mixed thoroughly, and then allowed to stand to hybridize the Y chain and the X chain, and then Lambda exonuclease is added for incubation for a period of time, and finally heated to 90°C to inactivate the Lambda exonuclease.

6. The quantitative detection method according to claim 1, wherein In step 4, the P chain is labeled with a fluorescent group at the 5' end and a quenching group at the 3' end.

7. The quantitative detection method according to claim 6, wherein The fluorescent group and the corresponding quenching group are selected from one of the following combinations: FAM and BHQ1 / DABCYL / TAMRA, TET and BHQ1 / DABCYL, HEX and BHQ1 / BHQ2 / DABCYL, TAMRA and BHQ2, ROX and BHQ2, Texas Red and BHQ2, Cy5 and BHQ2 / BHQ3.

8. The quantitative detection method according to claim 1, wherein The rate of increase of the fluorescence of the sample to be tested is measured from the curve of the change of the fluorescence intensity with time obtained in step 4, and the content of the base deletion sites in the sample to be tested is calculated according to the standard curve obtained by linear fitting of the rate of increase of the fluorescence and the concentration of the base deletion sites from the standard samples in step 5.

9. A quantitative detection kit for base deletion sites in genomic DNA sequences, comprising three oligonucleotide chains: X chain, Y chain and P chain, T4 PDGase and its working buffer, Lambda exonuclease and its working buffer; wherein, The X chain is an oligodeoxynucleotide chain with a hydroxylamine modification at the 5' end, and has a length of 14-16 nt; the Y chain can hybridize with the X chain to form a double-stranded DNA structure with 2 nucleotide mismatches at the 5' end of the X chain and the rest being matched; and the P chain is an oligonucleotide probe labeled with a fluorescent group and a quenching group at both ends, and can hybridize with the X chain to form a double-stranded DNA structure with 2 nucleotide mismatches at the 5' end of the P chain and the rest being matched.

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